Programmed ribosomal frameshifting is a specific mode of gene regulation designed to increase the informational content of small and limited viral genomes. Programmed ribosomal frameshifting in all viruses occurs by the same mechanism (Brierley, 1995, J. Gen. Virol., 76:1885-1892; Farabaugh, 1997, “Programmed alternative reading of the genetic code,” R.G. Landes Company, Austin, Tex.; Jacks, 1990, Curr. Top. Microbiol. Immunol. 157:93-124). Two basic RNA elements, a slippery site and downstream stem-loop structure, have been identified which are generally required to generate and regulate −1 ribosomal frameshifting (Brierley, 1995, J. Gen. Virol., 76:1885-1892; Farabaugh, 1997, “Programmed alternative reading of the genetic code,” R.G. Landes Company, Austin, Tex.; Bekaert, et al., 2003, Bioinformatics, 19:327-335; Dulude, et al., 2002, Nucleic Acids Res., 30:5094-5102; Gaudin, et al., 2005, J. Mol. Biol., 349:1024-1035; Staple, et al., 2005, J. Mol. Biol. 349:1011-1023). The slippery site consists of a stretch of seven nucleotides that do not have a uniform sequence but span three amino acid codons and must conform to the sequence X XXY YYZ (the gag ORF is indicated by spaces) where X is any nucleotide, Y is an A or U, and Z is A, U, or C (Brierley, et al., 1992, J. Mol. Biol. 227:463-479; Dinman, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:174-178; Dinman, et al., 1992, J. Virol. 66:3669-3676; Jacks, et al., 1988, Cell, 55:447-458). Earlier studies indicate that the downstream sequence forms a pseudoknot (Brierley, 1995, J. Gen. Virol, 76:1885-1892; Farabaugh, 1997, “Programmed alternative reading of the genetic code,” R.G. Landes Company, Austin, Tex.; Bekaert, et al., 2003, Bioinformatics, 19:327-335). However, recent data indicate that the downstream sequence forms a stem-loop structure (Dulude, et al., 2002, Nucleic Acids Res., 30:5094-5102; Gaudin, et al., 2005, J. Mol. Biol, 349:1024-1035; Staple, et al., 2005, J. Mol. Biol. 349:1011-1023). The stem-loop is a sequence that forms a defined RNA secondary structure and is thought to regulate production of the Gag-Pol polyprotein (Brierley, et al., 1989, Cell, 57:537-547; Dinman, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:174-178; Dulude, et al., 2002, Nucleic Acids Res., 30:5094-5102; Morikawa, et al., 1992, Virology, 186:389-397; Plant, et al., 2003, RNA, 9:168-174; Somogyi, et al., 1993, Mol. Cell. Biol. 13:6931-6940; Tu, et al., 1992, Proc. Natl. Acad. Sci. USA, 89:8636-8640). The importance of the stem loop in programmed ribosomal frameshifting is evidenced by the fact that human immunodeficiency virus-1 (HIV-1) genomes with mutations in the stem loop have shown a reduction in frameshift activity and have been found to be profoundly defective in viral replication (Telenti, et al., 2002, J. Virol. 76:7868-7873).
The rate of programmed ribosomal frameshifting is strictly regulated. Small changes in either or both the slippery site and stem-loop have been shown to have profound effects on the efficiency of ribosomal frameshifting (Baril, et al., 2003, RNA, 9:1246-1253; Brierley, 1995, J. Gen. Virol., 76:1885-1892; Dinman, 1995, Yeast, 11:1115-1127; Farabaugh, 1997, “Programmed alternative reading of the genetic code,” R.G. Landes Company, Austin, Tex.). The sequences of the RNA elements may affect the secondary structure and thermodynamic stability of the stem-loop. Furthermore, these sequences can affect the relative position of the RNA stem-loop in relation to the slippery site, in turn affecting the ability of the ribosome-bound tRNAs to unpair from the 0-frame codon, and thus the ability of these tRNAs to then pair with the −1 frame codon (Brierley, et al., 1989, Cell, 57:537-547; Brierley, et al., 1992, J. Mol. Biol. 227:463-479; Brierley, et al., 1991, J. Mol. Biol. 220:889-902; Dinman, et al., 1991, Proc. Natl. Acad. Sci. USA, 88:174-178; Dinman, et al, 1992, J. Virol. 66:3669-3676; Honda, et al, 1995, Biochem. Biophys. Res. Commun. 213:575-582; Jacks, et al., 1988, Cell, 55:447-458; Jacks, et al., 1988, Nature, 331:280-283; Morikawa, et al., 1992, Virology, 186:389-397; Namy, et al., 2006, Nature, 441:244-247).
Several viruses have been shown to utilize −1 programmed ribosomal frameshifting to produce the Gag-Pol polyprotein. Frameshifting ensures that the correct ratio of viral structural proteins (Gag) to non-structural proteins (Pol) is maintained in the cytoplasm (Brierley, 1995, J. Gen. Virol., 76:1885-1892). The maintenance of a precise ratio of Gag to Gag-Pol synthesis for viral propagation has been demonstrated for a number of retroviruses as well as for endogenous viruses of the yeast Saccharomyces cerevisiae (Dinman, et al., 1998, Trends Biotech. 16(4):3669-3676; Telenti, et al., 2002, J. Virol. 76:7868-7873). The ratio of HIV-1 Gag to Gag-Pol synthesized as a consequence of −1 programmed ribosomal frameshifting varies within a narrow window of approximately 10:1 to 20:1 (Farabaugh, 1997, “Programmed alternative reading of the genetic code,” R.G. Landes Company, Austin, Tex.; Jacks, et al., 1988, Nature, 331:280-283; Telenti, et al., 2002, J. Virol. 76:7868-7873). Changing the ratio of Gag to Gag-Pol proteins in retroviruses like HIV-1 or Moloney murine leukemia virus interferes with particle formation and replication (Farabaugh, 1997, “Programmed alternative reading of the genetic code,” R.G. Landes Company, Austin, Tex.; Felsenstein, et al., 1988, J. Virol. 62(6):2179-2182; Karacostas, et al., 1993, Virology, 193:661-671; Park, et al., 1991, J. Virol. 65(9):5111-5117). For example, overexpression of the Gag-Pol precursor protein results in the inefficient processing of the polyprotein and consequently inhibition of virus production. It has been shown that maintaining the proper ratio of Gag to Gag-Pol is necessary for proteolytic processing as well as for RNA dimerization (essential for the packaging of the viral RNA genome) and viral infectivity (Hill, et al., 2002, J. Virol. 76:11245-11253; Shehu-Xhilaga, et al., 2001, J. Virol. 75(4):1834-1841).
Clearly, alterations in programmed ribosomal frameshifting efficiencies can have a pronounced negative effect on viral production. Thus, ribosomal frameshifting is a compelling, unexploited target for novel antiviral agents (Dinman, et al., 1998, Trends Biotechnol. 16:190-196).
Accordingly, the present invention provides compounds capable of modulating the efficiency of programmed ribosomal frameshifting, methods by which such compounds capable of modulating the efficiency of viral programmed ribosomal frameshifting may be identified or validated and methods for treating viral infections using such compounds.